©2007 L

ANDES BIOSCI

ENCE.

DO NOT DIST

RIBUTE.

Review

Transcription Factor Networks in Embryonic Stem Cells and Testicular Cancer and the Definition of Epigenetics

[Epigenetics 2:1, 37-42, January/February/March 2007]; ©2007 Landes Bioscience

Wolfgang A. Schulz*Michèle J. Hoffmann

Department of Urology; Heinrich Heine University; Düsseldorf Germany

*Correspondence to: Wolfgang A. Schulz; Department of Urology; Heinrich Heine University; Moorenstr. 5; 40225 Düsseldorf Germany; Tel.: +49.211.81.18966; Fax: +49.211.81.15846; Email: wolfgang.schulz@uni-duesseldorf.de

Original manuscript submitted: 01/09/07Manuscript accepted: 02/27/07

Previously published online as an Epigenetics E-publication:http://www.landesbioscience.com/journals/epigenetics/abstract.php?id=4067

Key WordS

seminoma, teratocarcinoma, NANOG, OCT4, SOX2, chromatin, cell differen-tiation, transcription factor cascade, DNA methylation

AcKnoWledgeMentS

The authors wish to thank Dr. Jiri Hatina for stimulating discussions. Financial support by the Deutsche Krebshilfe and the Christiane und Claudia Hempel Stiftung is gratefully acknowledged.

AbStrActThe stem cell phenotype of human and murine ES cells has recently been shown to be

maintained by a self‑stabilizing network of transcription factors, NANOG, OCT4, and SOX2. These factors maintain their own and each other’s transcription, activating, by combinatorial interactions, genes responsible for the ES cell phenotype while repressing genes required for differentiation. This ‘core circuitry’ interacts with an ‘expanded circuitry’ encompassing signal transduction and chromatin regulator proteins. During ES cell differentiation the crucial transcription factors are down‑regulated by epigenetic mechanisms, including DNA methylation. Aberrant activation of the ES transcription factor network elicited by increased dosage of an embryonic gene cluster at 12p including NANOG, together with additional genetic and epigenetic alterations, appears to be a crucial event in the genesis of testicular germ cell cancers. Intriguingly, the ES cell transcription factor network fits current as well as past definitions of ‘epigenetic’.

Are trAnScription FActor netWorKS epigenetic?The notion of ‚epigenetics’ conceived by C.H. Waddington more than 50 years ago

was an attempt to conceptualize the phenotypic effects resulting from the complex inter-actions between multiple genes during embryonic development and cell differentiation (excellently reviewed in ref. 1). Initially, the concept inspired system theoreticians more than molecular biologists, although it may experience a revival in the present strife towards a “systems biology”. The notion ‘epigenetic’ that has become customary in the last decade is not so far-reaching. It refers to mechanisms which allow the propagation of properties of a cell (or an organism) without changes in the DNA sequence; a stricter usage requires such mechanisms to be nuclear. The prototypic mechanism fitting this definition is, of course, DNA methylation. Processes like histone methylation or changes in histone variants distinguishing active genes are also commonly encompassed under the heading of ‘epigenetics’. This is considered justified, because overall chromatin structure at individual genes can be inherited over many cell generations, even though individual histone modi-fications, especially acetylation, are too dynamic to be passed on between cell generations. Like DNA methylation, chromatin structure can be stably inherited, or can be switched to a different epigenetic state during cell differentiation or development. Chromatin structure is influenced by DNA methylation and vice versa. In addition, both are regulated by and, conversely, act upon transcription factors—repressors as well as activators.

Many instances of cell differentiation depend on transcription factors ‘cascades’. Textbook examples of such cascades include the transcription factor sets that act succes-sively during hematopoetic differentiation and muscle cell differentiation—a perhaps less well known example is hepatocyte differentiation.2,3 Transcription factors involved in cell differentiation induce the transcription of tissue-specific genes with concomitant changes in chromatin structure and sometimes DNA methylation. Importantly, they act upon each other within a network of interactions involving feedforward and feedback relationships that ensures stable and often irreversible cell differentiation (Fig. 1). Typically, in most cell lineages, the composition of the transcription factor set changes with each successive differentiation stage (in what was once termed ‘quantal’ cell cycles4) and the final state is terminal differentiation.

Transcription factor networks directing cell differentiation interact with epigenetic mechanisms including DNA methylation and histone modification. Likewise, the DNA sequence does not change during differentiation processes, except in certain inverte-brates and in the lymphocytes of mammals. So, usually a new phenotype is generated

www.landesbioscience.com Epigenetics 37

©2007 L

ANDES BIOSCI

ENCE.

DO NOT DIST

RIBUTE.

ES Transcription Factor Networks

Figure 1. A transcription factor cascade during cell differentiation. Cell differentiation takes place from top to bottom driven by successive activation of transcription factors (TF1–TF3) which act through specific response elements (TF1RE–TF3RE). TF3 also activates its own transcription in an autoregulatory feedforward loop. Tissue‑specific proteins (TSP1, TSP2) are induced by combinatorial action of the transcription factors. With increasing differentiation, proliferative potential decreases (arrows on the right), this is suggested to be regulated by TF2 and TF3. The transcription factor cascade shown in the figure is abstracted and much simplified compared to real cascades as found during differentiation of hematopoietic cells, myoblasts or hepatocytes.

38 Epigenetics 2007; Vol. 2 Issue 1

©2007 L

ANDES BIOSCI

ENCE.

DO NOT DIST

RIBUTE.

ES Transcription Factor Networks

without changes in the DNA sequence, in accord with the present day definition of ‘epigenetic’. Nevertheless, strictly speaking, such transcription factor networks cannot be considered ‚epigenetic’, since the state of the cells changes at each differentiation step along with the composition of the transcription factor network itself (Fig. 1) and the cell phenotype is thus not stably maintained (only the lineage). Intriguingly, such changes correspond to the original concept of ‘epigenetics’ aimed at explaining gene interactions during differentiation and development.

The issue is evidently much more straightforward in the case of stem cells. Obviously, as their DNA content does not differ from that of more differentiated cells, their stably propagated state must be maintained by epigenetic mechanisms. These presumably include specific and stably propagated DNA methylation patterns and chromatin structure. For instance, DNA methylation profiles were reported to distinguish human ES cells from other cell types, including 24 cancer cell lines, adult stem cells, lymphoblastoid cell lines, normal human tissues, and an embryonic carcinoma line. Relevant differences in methylation concern CpG-rich loci, known tumor suppressor genes, the MHC region, and methylcytosine levels at large.5 Likewise, the chromatin structure of ES cells is apparently distinctive.6,7 In addition, several recent papers have indicated that a crucial determinant for the stable and unlimited propagation of embryonic stem cells is a network (or ‘circuitry’) of interacting transcription factors. These factors mutually and autocatalytically maintain their own expression and repress genes required for differentiation, directly or through interaction with other factors, including established epigenetic regulators (Fig. 2). By all criteria, this self-maintaining network of (nuclear) transcription factors fits any definition of ‘epigenetic’.

trAnScription FActor netWorKS in SteM cellSThe identification of this transcription factor network culminated

in two papers8,9 on human and mouse ES cells, respectively. They extend previous and concurrent work10-13 elucidating in particular

the interaction between three transcription factors, viz. NANOG, OCT4 (alias POU5F1), and SOX2, during embryonic and germ cell development. In human ES cells, chromatin immunoprecipita-tion (ChIP) identified 623 gene promoters bound by OCT4, 1271 bound by SOX2, and 1687 occupied by NANOG. In addition to these protein-encoding genes, several genes binding one or several of the three embryonal transcription factors encode miRNAs. The target gene sets for the factors overlapped considerably, with 35% of the target genes binding two factors, and 20% all three. Importantly, the three factors regulate each other and themselves positively, forming feedforward loops. Accordingly, an independent detailed study10 showed reciprocal transcriptional regulation of Oct4 and Sox2 by Sox/Oct complexes in ES cells and combinatorial binding of the two factors to the enhancers of both Sox2 and Oct4 genes. In that study, Sox and Oct4 were found to interact with the Nanog promoter and suggested to act upstream in the hierarchy. Conversely, a study in human gonocytes placed NANOG upstream of OCT4.11 It is possible that these differences have a biological rather than an experimental basis, but most likely they reflect a general property of robust self-maintaining networks, namely that they can be activated by stimulating any of the crucial factors involved.

NANOG, SOX2, and OCT4 can act as transcriptional repressors as well as activators. Positive target genes of the factors identified in ref. 8 contain several others that are strongly expressed in ES cells and supposed to contribute to pluripotency and self-renewal. Repressed targets include many genes that are known to become activated during and contribute to the differentiation of ES cells. Interestingly, several target genes are chromatin regulators, e.g., the remodelling protein SMARCAD1, the histone acetylase MYST3, and a SET histone methylase (Fig. 2). Moreover, the polycomb protein Suz12 was found to occupy and repress target genes together with OCT4, SOX2 and NANOG.12 Another group of positive targets encodes transcription factors, in particular STAT3, a crucial positive regulator of mouse ES cell renewal and the ultimate target of LIF, and REST, a repressor of neuronal differentiation.15 On a note of caution, cultured human ES cells may not be dependent on LIF.16 Besides STAT signaling, components of the TGFb signal transduc-tion pathway, which is likewise essential for ES cell maintenance, were conspicuous. The authors designated these additional factors, chromatin regulators and signaling pathways, the ‘expanded tran-scriptional circuitry’ of ES cells (Fig. 2). The expanded circuitry was also evident in an independent study, in which OCT4 siRNA altered expression not only of OCT4 itself, NANOG, and SOX2, but also of many genes involved in epigenetic regulation, chromatin remodel-ling, apoptosis and signal transduction.13 For instance, this study revealed the WNT antagonist DKK1 (Dickkopf ) as a target of the ES transcription factor network.

In the study on mouse ES cells,9 a different ChIP-technique (ChIP- PET) was employed, which aims at identifying as well more remote binding sites for transcription factors. This study focussed initially on detecting regions in the genome that bind Oct4 as well as Nanog. In addition, in-depth experiments were performed using siRNA against the transcription factors to ascertain their effects on selected genes and to define the Nanog binding sites more precisely. The study confirmed that both factors have many common target genes. However, a relative small fraction of the genes identified in this study (fewer than 13%) were identical with those in the study on human ES cells, especially if only promoter binding was consid-ered. Crucially, the core autoregulatory circuit from human cells was also evident in mouse ES cells, with Oct4 and Nanog binding

Figure 2. Regulation of self‑renewal and differentiation in ES cells. The transcription factors constituting the ‘core circuitry’ activate each other’s and their own transcription. They promote stem cell self‑renewal through STAT3 and TGFb signaling proteins (TGFb‑SP) and block differentiation. All func‑tions (maintenance of the network, promotion of self‑renewal, and inhibition of differentiation) are supported by interactions with chromatin regulators of the ‘expanded circuitry’.

www.landesbioscience.com Epigenetics 39

©2007 L

ANDES BIOSCI

ENCE.

DO NOT DIST

RIBUTE.

ES Transcription Factor Networks

to their own and mutual promoters as well as to the Sox2 promoter (in accord with ref. 10). Moreover, several components of the ‘expanded’ transcriptional network of human cells were also discovered in mouse cells, especially Rest and Smarcad1. While several plausible targets from the human study were missing, several others were identified that one might have been expected in human cells too, such as Mycn, Tp53, and Dkk1. Interestingly, among 32 gene promoters identified as common targets of Oct4 and Nanog in mouse and human ES cells, 18 are transcriptional regulators (in a narrow sense). This provides a solid basis for the denomination ‘transcription factor network’.

epigenetic inActivAtion oF SteM cell MAintenAnce FActorS during diFFerentiAtion

During differentiation of ES cells, various transcription factor cascades directing cell differentiation become activated, with initiating factors released from the repression by the core and extended transcriptional circuitry of the stem cells. Conversely, the crucial stem cell maintainance factors become irreversibly downregulated. Again, because this process does not involve changes in the DNA sequence, the loss of the stem cell phenotype must be regulated by epigenetic mechanisms. For instance, the Oct4/OCT4 promoter becomes methylated in mouse and human cells during development17,18 and its methylation in somatic human cells is remarkably stable, even in cancer cell lines with otherwise strongly hypomethylated DNA.18 The epigenetic inactivation of OCT4 requires chromatin regulators like the histone methyltransferase G9a,19 which interacts with the EZH2 polycomb protein. Conversely, in ES cells polycomb proteins are associated with OCT4, SOX2, and NANOG targets, but are released upon differentiation.12 So, during differentiation of ES cells, the core transcriptional circuitry is epigenetically inactivated and the accessory chromatin regulators from the expanded circuitry appear to move to different targets.

The importance of the epigenetic inactivation of the ES cell transcription factor circuitry is illustrated by experiments in which somatic cells are fused with ES cells (reviewed in ref. 20). These fusions can lead to pluripotent stem cells and crucially depend on the activity of Nanog. They can be monitored by an Oct4 transgene in which Oct4 regulatory elements drive a reporter gene in the somatic cell partner that becomes reactivated upon fusion.21 Like the Oct4 gene, other genes and regions inactivated in somatic cells become remodelled and revert to the embryonic state. Even the inactive X chromosome loses its epigenetic histone modifications. Likewise, signal transduction pathways characteristic of ES cells become dominant. Thus, in addition to the core circuitry, the expanded circuitry dominates in the fusion cells.

iMplicAtionS For teSticulAr gerM cell cAncerSThese findings have direct and important implications for the

understanding of an important group of diseases, viz. testicular germ cell cancers. Testicular germ cell cancers have an unusual age distribu-tion and represent the most frequent cancer type in young men in the third and fourth decade of life, especially in Europe. Although overall rare, there is a general increase in their incidence, which is alarming in some countries like Denmark, Switzerland, and the Czech Republic, where life-time risks meanwhile approach 1%.22 While the causes of this increase are hotly debated (see ref. 23 for a popular account), it is generally agreed that the initiation of testicular cancers occurs during intrauterine development. It is assumed that the different histological types of testicular cancers, seminoma, embryonal

carcinoma, teratocarcinoma, and yolk sac tumors derive from primary germ cells arrested in their development at a premeiotic stage.24,25 The germ cell cancers found in extragonodal sites in children are thought to derive from even earlier stages of the same developmental lineage, and the spermatocytic seminomas occuring in older men from more differentiated cells. Likely, intrauterine carcinogenesis leads to an obligate precursor lesion, intratubular germ cell neoplasia (ITGCN, also named ‘carcinoma in situ testis’), from which cancer develops after puberty by accumulation of additional genetic alterations. The initiating (genetic or epigenetic) event for testicular germ cell cancers is not precisely defined, but approximately 90% of the cases share a common chromosomal aberration, isochromosome 12p. This leads obviously to an increased dosage of genes on the short arm of the chromosome as well as to a decreased dosis of genes on the long arm. Since many of the remaining 10% of testicular germ cell cancers harbor amplifications of parts of 12p, the former change is considered more important.

Human embryonal carcinoma (EC) cell lines show expression profiles very similar to those of human ES cells.26 The genes upregulated in EC lines comprise OCT4, the de novo DNA methyl- transferase DNMT3B, the transcription factor FOXD3, and the WNT receptor FRZ7. These genes were also found expressed in cancer tissues. Many, including OCT4 were also found in semi-noma tissues, but expression profiles in seminomas were clearly more different from ECs than those in ES cells. Overexpression of POU5F1/OCT4 had been previously reported to provide an excellent immunohistochemical marker for testicular cancers. In particular, OCT4 expression characterized the stem cell fraction in teratocarcinomas,27 which consist of a cancerous embryonal carcinoma-like cell population and differentiated, often non-malig-nant components.These findings fit the concept that seminomas are a precursor stage of embryonal carcinomas that is more closely related to primordial germ cells. Embryonal carcinomas derive from seminomas by developing towards a stage resembling the ICM from which ES cells are derived. Evidently, teratocarcinomas develop from embryonal carcinomas, by acquiring the ability to partially differen-tiate into various somatic lineages while retaining a stem cell fraction composed of EC cells. Interestingly, the development of teratocarci-nomas from EC can be traced as well by the accumulation of DNA hypermethylation at a number of genes, which is very rare in EC.28

Sperger et al.26 noticed that many of the genes characterizing the EC expression profile were located on chromosome 12. This conclusion was strengthened by a further study of the undifferentiated component of embryonal carcinoma tissues and cell lines that identified OCT4 and NANOG as overexpressed, alongside DNMT3B, the male germ cell-specific DNMT3L, and components of the TGFb, Notch and WNT signaling pathways.29 The WNT and Notch signaling pathways are downregulated during retinoic acid-induced differentiation of ES and EC cell lines.30 In addi-tion to NANOG, several other genes in an ‘embryonic cluster’ on chromosome 12p, like GDF3, were consistently overexpressed in cancers compared to normal testis tissue. These might be considered part of the “extended” ES circuitry as defined by Boyer et al.8 Genes characteristic of seminoma included the SCF receptor tyrosine kinase receptor KIT, already well established as involved in human testicular cancer25,31-33 and the embryonic ‘antigen’ PRAME, a repressor of retinoic acid signaling. The 12p13.3 gene cluster was studied in detail more recently.34 NANOG, STELLA, and the uncharacterized gene Hs.129302 were found to be strongly overexpressed in seminomas as well as EC, whereas GDF3 was more variable in seminomas. All four genes showed lower or more variable expression in teratocarcinomas and other rarer more differentiated tumor types.

40 Epigenetics 2007; Vol. 2 Issue 1

©2007 L

ANDES BIOSCI

ENCE.

DO NOT DIST

RIBUTE.

ES Transcription Factor Networks

Thus, the transcription factor circuitry characteristic of ES cells is retained active in human germ cell cancers and, likely, the inability of the cancer precursor cells to inactivate this network represents a crucial carcinogenic event. The mechanism underlying this failure is very likely related to the altered dosage of NANOG and its neighboring genes. Whether this dosage change is sufficient to block the developmental downregulation of the ES transcrip-tion factor network, remains to be determined. Conceivably, other alterations may contribute to stabilizing the ES circuitry in testicular cancers and to the development arrest of their precursors (Fig. 3). For instance, cultured human ES cells gain chromosome 17 in addition to chromosome 12, as do some EC. Chromosome 17 among others harbors the GRB7, Survivin, STAT3, HER2, and GRB2 genes, whose increased expression may favor self-renewal and survival over differentiation.35 In rare patients with bilateral cancers, mutations in the KIT tyrosine receptor kinase are likely to constitute an initiating event in carcinogenesis.

KIT and its ligand stem cell factor (SCF) are definitely important in testicular cancers, as they are for the survival of normal germ cell precursors, but neither mutations nor gene amplification are common outside the group of patients with bilateral diseases. Therefore, the initiating event leading to testicular intratubular neoplasia remains uncertain in most cases. The aberrant maintenance of an active state of the newly discovered embryonal epigenetic transcription factor network is obviously necessary for the development of testicular cancers, but the underlying genetic change, alteration of chromo-some 12p, has not been detected in all precursors. So, might the initiating event be epigenetic? This idea is certainly speculative, but evidence for altered imprinting in a subset of childhood extragonodal cancers has been provided.36 In conclusion, nevertheless, while the initiation mechanism of testicular germ cell cancers remains to be fully elucidated, through the identification of an epigenetic transcrip-tion factor network in embryonic cells one essential factor in the development of these cancers appears to have been identified.

References 1. Slack JM. Conrad hal waddington: The last Renaissance biologist? Nat Rev Genet 2002;

3:889-95. 2. Costa RH, Kalinichenko VV, Holterman AX, Wang X. Transcription factors in liver devel-

opment, differentiation, and regeneration. Hepatology 2003; 38:1331-47. 3. Odom DT, Dowell RD, Jacobsen ES, Nekludova L, Rolfe PA, Danford TW, Gifford DK,

Fraenkel E, Bell GI, Young RA. Core transcriptional regulatory circuitry in human hepato-cytes. Mol Syst Biol 2006; 2:2006.

4. Holtzer H, Rubinstein N, Fellini S, Yeoh G, Chi J, Birnbaum J, Okayama M. Lineages, quantal cell cycles, and the generation of cell diversity. Q Rev Biophys 1975; 8:523-57.

5. Bibikova M, Chudin E, Wu B, Zhou L, Garcia EW, Liu Y, Shin S, Plaia TW, Auerbach JM, Arking DE, Gonzalez R, Crook J, Davidson B, Schulz TC, Robins A, Khanna A, Sartipy P, Hyllner J, Vanguri P, Savant-Bhonsale S, Smith AK, Chakravarti A, Maitra A, Rao M, Barker DL, Loring JF, Fan JB. Human embryonic stem cells have a unique epigenetic sig-nature. Genome Res 2006; 16:1075-83.

6. Martens JH, O’Sullivan RJ, Braunschweig U, Opravil S, Radolf M, Steinlein P, Jenuwein T. The profile of repeat-associated histone lysine methylation states in the mouse epigenome. EMBO J 2005; 24:800-12.

7. Azuara V, Perry P, Sauer S, Spivakov M, Jorgensen HF, John RM, Gouti M, Casanova M, Warnes G, Merkenschlager M, Fisher AG. Chromatin signatures of pluripotent cell lines. Nat Cell Biol 2006; 8:532-8.

8. Boyer LA, Lee TI, Cole MF, Johnstone SE, Levine SS, Zucker JP, Guenther MG, Kumar RM, Murray HL, Jenner RG, Gifford DK, Melton DA, Jaenisch R, Young RA. Core tran-scriptional regulatory circuitry in human embryonic stem cells. Cell 2005; 122:947-56.

9. Loh YH, Wu Q, Chew JL, Vega VB, Zhang W, Chen X, Bourque G, George J, Leong B, Liu J, Wong KY, Sung KW, Lee CW, Zhao XD, Chiu KP, Lipovich L, Kuznetsov VA, Robson P, Stanton LW, Wei CL, Ruan Y, Lim B, Ng HH. The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells. Nat Genet 2006; 38:431-40.

10. Chew JL, Loh YH, Zhang W, Chen X, Tam WL, Yeap LS, Li P, Ang YS, Lim B, Robson P, Ng HH. Reciprocal transcriptional regulation of Pou5f1 and Sox2 via the Oct4/Sox2 complex in embryonic stem cells. Mol Cell Biol 2005; 25:6031-46.

11. Rodda DJ, Chew JL, Lim LH, Loh YH, Wang B, Ng HH, Robson P. Transcriptional regula-tion of nanog by OCT4 and SOX2. J Biol Chem 2005; 280:24731-7.J Biol Chem 2005; 280:24731-7.

12. Lee TI, Jenner RG, Boyer LA, Guenther MG, Levine SS, Kumar RM, Chevalier B, Johnstone SE, Cole MF, Isono K, Koseki H, Fuchikami T, Abe K, Murray HL, Zucker JP, Yuan B, Bell GW, Herbolsheimer E, Hannett NM, Sun K, Odom DT, Otte AP, Volkert TL, Bartel DP, Melton DA, Gifford DK, Jaenisch R, Young RA. Control of developmental regulators by Polycomb in human embryonic stem cells. Cell 2006; 125:301-13.

13. Babaie Y, Herwig R, Greber B, Brink TC, Wruck W, Groth D, Lehrach H, Burdon T, Adjaye J. Analysis of OCT4 dependent transcriptional networks regulating self renewal and pluripotency in human embryonic stem cells. Stem Cells 2006; 25:500-10.

14. Hoei-Hansen CE, Almstrup K, Nielsen JE, Brask SS, Graem N, Skakkebaek NE, Leffers H, Rajpert-De Meyts E. Stem cell pluripotency factor NANOG is expressed in human fetal gonocytes, testicular carcinoma in situ and germ cell tumours. Histopathology 2005; 47:48-56.

15. Westbrook TF, Martin ES, Schlabach MR, Leng Y, Liang AC, Feng B, Zhao JJ, Roberts TM, Mandel G, Hannon GJ, Depinho RA, Chin L, Elledge SJ. A genetic screen for candidate tumor suppressors identifies REST. Cell 2005; 121:837-48.

16. Daheron L, Opitz SL, Zaehres H, Lensch WM, Andrews PW, Itskovitz-Eldor J, Daley GQ. LIF/STAT3 signaling fails to maintain self-renewal of human embryonic stem cells. Stem Cells 2004; 22:770-8.

17. Gidekel S, Bergman Y. A unique developmental pattern of Oct-3/4 DNA methylation is controlled by a cis-demodification element. J Biol Chem 2002; 277:34521-30.

18. Hoffmann MJ, Müller M, Engers R, Schulz WA. Epigenetic control of CTCFL/BORIS and OCT4 expression in urogenital malignancies. Biochem Pharmacol 2006; 72:1577-88.

19. Feldman N, Gerson A, Fang J, Li E, Zhang Y, Shinkai Y, Cedar H, Bergman Y. G9a-mediated irreversible epigenetic inactivation of Oct-3/4 during early embryogenesis. Nat Cell Biol 2006; 8:188-94.

20. Tada T. Toti-/pluripotential stem cells and epigenetic modifications. Neurodegener Dis 2006; 3:32-7.

21. Silva J, Chambers I, Pollard S, Smith A. Nanog promotes transfer of pluripotency after cell fusion. Nature 2006; 441:997-1001.

22. Bray F, Richiardi L, Ekbom A, Pukkala E, Cuninkova M, Moller H. Trends in testicular cancer incidence and mortality in 22 European countries: Continuing increases in incidence and declines in mortality. Int J Cancer 2006; 118:3099-111.

23. Cadbury D. Altering Eden: The feminization of nature. London: Penguin Books Ltd., 1998.

24. Oosterhuis JW, Looijenga LH. Testicular germ-cell tumours in a broader perspective. Nat Rev Cancer 2005; 5:210-22.

25. Reuter VE. Origins and molecular biology of testicular germ cell tumors. Mod Pathol 2005; 18:S51-S60.

26. Sperger JM, Chen X, Draper JS, Antosiewicz JE, Chon CH, Jones SB, Brooks JD, Andrews PW, Brown PO, Thomson JA. Gene expression patterns in human embryonic stem cells and human pluripotent germ cell tumors. Proc Natl Acad Sci USA 2003; 100:13350-5.

27. de Jong J, Stoop H, Dohle GR, Bangma CH, Kliffen M, van Esser JW, van den BM, Kros JM, Oosterhuis JW, Looijenga LH. Diagnostic value of OCT3/4 for pre-invasive and inva-sive testicular germ cell tumours. J Pathol 2005; 206:242-9.

Figure 3. Activation of the ES cell transcription factor network in testicular germ cell cancers. The stem cell phenotype of testicular germ cell cancers is apparently maintained by very similar mechanisms as that of ES cells. The activation of the ES transcription factor network appears to be caused by isochromosome 12p formation leading to an increased NANOG dosage together with additional genetic aberrations that are suggested here to act primarily on the expanded circuitry.

www.landesbioscience.com Epigenetics 41

©2007 L

ANDES BIOSCI

ENCE.

DO NOT DIST

RIBUTE.

ES Transcription Factor Networks

28. Smiraglia DJ, Szymanska J, Kraggerud SM, Lothe RA, Peltomaki P, Plass C. Distinct epi-genetic phenotypes in seminomatous and nonseminomatous testicular germ cell tumors. Oncogene 2002; 21:3909-16.

29. Skotheim RI, Lind GE, Monni O, Nesland JM, Abeler VM, Fossa SD, Duale N, Brunborg G, Kallioniemi O, Andrews PW, Lothe RA. Differentiation of human embryonal carcino-mas in vitro and in vivo reveals expression profiles relevant to normal development. Cancer Res 2005; 65:5588-98.

30. Looijenga LH, de Leeuw H, van Oorschot M, van Gurp RJ, Stoop H, Gillis AJ, Gouveia Brazao CA, Weber RF, Kirkels WJ, van Dijk T, von Lindern M, Valk P, Lajos G, Olah E, Nesland JM, Fossa SD, Oosterhuis JW. Stem cell factor receptor (c‑KIT) codon 816 mutations predict development of bilateral testicular germ-cell tumors. Cancer Res 2003; 63:7674-8.

30. Walsh J, Andrews PW. Expression of Wnt and Notch pathway genes in a pluripotent human embryonal carcinoma cell line and embryonic stem cell. APMIS 2003; 111:197-210.

31. Kemmer K, Corless CL, Fletcher JA, McGreevey L, Haley A, Griffith D, Cummings OW, Wait C, Town A, Heinrich MC. KIT mutations are common in testicular seminomas. Am J Pathol 2004; 164:305-13.

32. Rapley EA, Hockley S, Warren W, Johnson L, Huddart R, Crockford G, Forman D, Leahy MG, Oliver DT, Tucker K, Friedlander M, Phillips KA, Hogg D, Jewett MA, Lohynska R, Daugaard G, Richard S, Heidenreich A, Geczi L, Bodrogi I, Olah E, Ormiston WJ, Daly PA, Looijenga LH, Guilford P, Aass N, Fossa SD, Heimdal K, Tjulandin SA, Liubchenko L, Stoll H, Weber W, Einhorn L, Weber BL, McMaster M, Greene MH, Bishop DT, Easton D, Stratton MR. Somatic mutations of KIT in familial testicular germ cell tumours. Br J Cancer 2004; 90:2397-401.

34. Korkola JE, Houldsworth J, Chadalavada RS, Olshen AB, Dobrzynski D, Reuter VE, Bosl GJ, Chaganti RS. Down-regulation of stem cell genes, including those in a 200-kb gene cluster at 12p13.31, is associated with in vivo differentiation of human male germ cell tumors. Cancer Res 2006; 66:820-7.

35. Draper JS, Smith K, Gokhale P, Moore HD, Maltby E, Johnson J, Meisner L, Zwaka TP, Thomson JA, Andrews PW. Recurrent gain of chromosomes 17q and 12 in cultured human embryonic stem cells. Nat Biotechnol 2004; 22:53-4.

36. Schneider DT, Schuster AE, Fritsch MK, Hu J, Olson T, Lauer S, Gobel U, Perlman EJ. Multipoint imprinting analysis indicates a common precursor cell for gonadal and nongo-nadal pediatric germ cell tumors. Cancer Res 2001; 61:7268-76.

42 Epigenetics 2007; Vol. 2 Issue 1